Unexpressable protein

Peter Ellis via methods%40net.bio.net (by pjie2 from cam.ac.uk)
Fri Sep 24 14:28:19 EST 2010


The protein I'm working on doesn't want to express in any culture or in 
vitro system I've tried.  So far I've been through:

pEXT5-CT vector (T7 promoter, C-terminal His tag).  No soluble or 
insoluble expression in BL21DE3 cells with autoinduction or with IPTG 
induction.  No soluble or insoluble expression using coupled T7 
transcription/translation systems, either with E. coli extract or 
reticulocyte lysate.

pT7CFE1 vector (T7 promoter, C terminal His tag, ribosome entry site and 
3'UTR optimised for use in vitro transcription/translation with 
mammalian extracts).  No soluble or insoluble expression with 
reticulocyte lysate transcription/translation system.

pGEX(KG) vector (tac promoter, N terminal GST fusion).  No soluble or 
insoluble expression using autoinduction or IPTG induction.

pEGFP-N1 vector (CMV promoter, N terminal GFP fusion).  No soluble or 
insoluble expression in two different mammalian cell lines.

All four of the above systems have worked fine in our hands with control 
genes (and some other genes I'm working on), so it's nothing wrong with 
the kits/cells or our lab technique.  For the latest attempt at in vitro 
transcription/translation, we isolated the mRNA from the reaction and 
sequenced it, so we know the constructs are being expressed and is 
in-frame etc. The protein just doesn't want to translate, no matter what 
system we use.

I guess my protein of interest just really doesn't like being expressed 
in culture!  Maybe it needs particular cofactors / chaperones in order 
to be translated?  Does anyone have any suggestions what to try next? 
The protein itself is about 50kDa, specific to round spermatids in the 
testis.  Spermatids are pretty weird cells, so it's certainly possible 
that my protein isn't happy in any other cell type. Unfortunately round 
spermatids don't grow in culture, so transfecting spermatids directly 
isn't an option.

About all I can think to try is to add some testis extract to an in 
vitro transcription/translation reaction and hope it supplies whatever 
the missing factors are.  Has anyone tried anything like that before, 
and is it a remotely sensible thing to try?  If so, what's the best way 
to go about it - homogenise testis tissue in RIPA buffer or PBS and just 
add some to the reaction?

Peter Ellis

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